Novel bacteriophage and antibacterial composition comprising the same

ABSTRACT

The present invention relates to a novel bacteriophage having a specific bactericidal activity against  salmonella , a composition for the prevention or treatment of infectious diseases comprising the bacteriophage as an active ingredient, an antibiotic comprising the bacteriophage as an active ingredient, an animal feed or drinking water comprising the bacteriophage as an active ingredient, and a sanitizer or cleaner comprising the bacteriophage as an active ingredient. The novel bacteriophage of the present invention has a specific bactericidal activity against  Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis  or  Salmonella newport  with no influences on beneficial bacteria, as well as excellent acid- and heat-resistance and desiccation tolerance. Therefore, the novel bacteriophage can be used for the prevention or treatment of salmonellosis or  salmonella  food poisoning, which is an infectious disease caused by  Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis  or  Salmonella newport , and also widely used in animal feeds, drinking water for livestock, sanitizers, and cleaners.

TECHNICAL FIELD

The present invention relates to a novel bacteriophage and anantibacterial composition comprising the same.

BACKGROUND ART

Salmonella has an average genomic GC content of 50-52%, which is similarto that of Escherichia coli and Shigella. The genus Salmonella is apathogenic microorganism that causes infections in livestock as well asin humans. Serological division has it that Salmonella enterica, aspecies of Salmonella bacterium, has a variety of serovars includingSalmonella gallinarum, Salmonella pullorum, Salmonella typhimurium (ST),Salmonella enteritidis (SE), Salmonella typhi, Salmonella choleraesuis(SC), Salmonella derby (SD). Of them, choleraesuis and derby areswine-adapted pathogens, gallinarum and pullorum are fowl-adaptedpathogens, typhimurium and enteritis are pathogenic for humans andanimals, and typhi is a human-adapted pathogen, all of which causeillness in their respective species, resulting in tremendous damage tofarmers and consumers (Zoobises Report; United Kingdom 2003).

Recently, implementation of HACCP (Hazard analysis and critical controlpoints) has become a mandatory requirement for all slaughterhouses inKorea as of Jul. 1, 2003, because of the high risk of contamination inthe course of manufacturing livestock products with salmonella, whichcauses direct damage to pigs, and meat hygiene and is found in thedigestive tract of pigs (Jae-gil Yeh. Characterization and Counterplanof Salmonellosis in Pigs. Monthly Magazine of Pig Husbandry. 2004).

Paratyphoid, the acute or chronic infectious disease in the digestivetract of pig caused by salmonella infection, is characterized bygastroenteritis and septicaemia and mainly occurs during the fattingperiod. In particular, some of pathogenic bacteria that cause thisdisease can cause food poisoning in humans through meat ingestion, andthus it is a disease having major public health importance. A variety oftypes of salmonella bacteria can be pathogenic. Among them, Salmonellacholeraesuis and Salmonella typhisuis known to cause hog cholera are themajor causes of acute salmonella septicaemia. Acute enteritis occursduring the fattening period, and is accompanied by irregular appetite,severe watery diarrhea, high fever, loss of vitality, pneumonia, andnervous signs. Discoloration of the skin may occur in some severe cases.Salmonella typhimurium, Salmonella enteritidis, and Salmonella derby arethe major causes of chronic enteritis.

Salmonellosis is caused by oral route through feed or water contaminatedwith salmonella, and thus these routes should be prevented. Contaminatedfeed, raw materials or water, or adult pigs carrying the pathogen can bemajor sources of infection. During the acute period of infection, pigsshed up to 10⁶ Salmonella choleraesuis or 10⁷ Salmonella typhimurium pergram of feces. However, many experimental infections reported successfuldisease reproduction with a dose of 10⁸ to 10¹¹ Salmonella. In anexperiment injecting 10³ Salmonella into pigs, the injected pigs showedno symptoms of the disease, but other pigs raised in the same pen showedtypical clinical symptoms. These results indicate that a large amount ofsalmonella grow in naturally infected pigs, resulting in the infectionof other pigs (Jung-Bok Lee. Control of the Recent Outbreaks of PorcineSalmonella and Proliferative Enteropathy. Korea Swine Association.2009).

At present, severe viral infections such as Porcine Reproductive andRespiratory Syndrome (PRRS) and Porcine Circovirus (PCV2) have beencausing tremendous economic losses to the swine industry in Korea, andthus disease management has been focused on these diseases. Since thesebacterial diseases may cause tremendous damage comparable to that causedby viral diseases beginning with a ban on the use of in-feed antibioticsand an investigation of disease occurrence or disease management shouldbe performed in advance (Jung-Bok Lee. Control of the Recent Outbreaksof Porcine Salmonella and Proliferative Enteropathy, Infectious DiseaseLaboratory, College of Veterinary Medicine, Konkuk University, LivestockProduct Safety, 2010) (Robert W. Wills, Veterinary Microbiology, 1999).Meanwhile, bacteriophage, also called phage, is a specialized type ofvirus that infects only particular bacteria and controls the growth ofbacteria, and can self-replicate only inside the host bacteria. Afterthe discovery of bacteriophages, a great deal of faith was initiallyplaced in their use for infectious-disease therapy. However, when broadspectrum antibiotics came into common use, bacteriophages were seen asunnecessary due to a specific target spectrum. Antibiotics orantimicrobial agents have been widely used for the treatment ofinfectious diseases caused by bacterial infection. Nevertheless, themisuse and overuse of antibiotics resulted in rising concerns aboutantibiotic resistance and the harmful effects of residual antibiotics infoods. However, the removal of current in-feed antibiotics mightincrease occurrence of bacterial diseases including salmonellosis thathave been controlled by antibiotics, as expected in the experiment dataor from other countries. Thus, there is an urgent need to establish adetailed practical guideline for salmonella management (Jung-Bok Lee.Control of the Recent Outbreaks of Porcine Salmonella and ProliferativeEnteropathy, Infectious Disease Laboratory, College of VeterinaryMedicine, Konkuk University, Livestock Product Safety. 2010).

These growing concerns have led to a resurgence of interest inbacteriophage. Seven bacteriophages for control of E. coli O157:H7 aredisclosed in U.S. Pat. No. 6,485,902 (2002) and two bacteriophages forcontrol of various microorganisms are disclosed in U.S. Pat. No.6,942,858 (issued to Nymox in 2005). Many companies have been activelytrying to develop various products using bacteriophages. EBI food system(Europe) developed a food additive for preventing food poisoning causedby Listeria monocytogenes, named Listerix-P100, which is the firstbacteriophage product approved by the USFDA. A phage-based product,LMP-102 was also developed as a food additive against Listeriamonocytogenes, approved as GRAS (Generally Regarded As Safe). In 2007, aphage-based wash produced by OmniLytics was developed to prevent E. coli0157 contamination of beef during slaughter, approved by USDA's FoodSafety and Inspection Service (FSIS). In Europe, Clostridium sporogenesphage NCIMB 30008 and Clostridium tyrobutiricum phage NCIMB 30008 wereregistered as feed preservative against Clostridium contamination offeed in 2003 and 2005, respectively. Such studies show that researchinto bacteriophages for the control of antibiotic-unsusceptible bacteriaand contamination of livestock products by zoonotic pathogens ispresently ongoing.

However, most of the phage biocontrol studies have focused on thecontrol of E. coli, Listeria, and Clostridium. Salmonella is also azoonotic pathogen, and damages due to this pathogen are not reduced. Asmentioned above, since Salmonella exhibits multiple drug resistances,nationwide antimicrobial resistance surveillance has been conducted inKorea under the Enforcement Decree of the Act on the Prevention ofContagious Disease (Executive Order 16961), Enforcement Ordinance of theAct on the Prevention of Contagious Disease (Ministry of Health andWelfare's Order 179), and Organization of the National Institute ofHealth (Executive Order 17164). Accordingly, there is a need for thedevelopment of bacteriophages to control Salmonella.

DISCLOSURE Technical Problem

In order to overcome problems occurring upon the misuse or overuse ofbroad spectrum antibiotics, such as drug resistant bacteria and drugresidues in foods, the present inventors isolated a bacteriophage fromnatural sources, in which the bacteriophage has a specific bactericidalactivity against salmonella causing major diseases in livestock. As aresult, they found that the bacteriophage has a specific bactericidalactivity against Salmonella choleraesuis (SC), Salmonella typhimurium(ST), Salmonella derby (SD), Salmonella infantis (SI) and Salmonellanewport (SN) with no influences on beneficial bacteria, in addition toshowing excellent acid- and heat-resistance, as identified for themorphological, biochemical and genetic properties thereof. Further, theyfound that the bacteriophage can be applied to compositions for theprevention or treatment of Salmonella typhimurium-mediated diseases,such as livestock salmonellosis and Salmonella food poisoning, and tovarious products for the effective prevention and control of Salmonellabacteria proliferation, including livestock feed additives, drinkingwater for livestock, barn sanitizers, and cleaners for meat products,thereby completing the present invention.

Technical Solution

An object of the present invention is to provide a novel bacteriophagehaving a bactericidal activity against Salmonella choleraesuis.

Another object of the present invention is to provide a composition forthe prevention or treatment of infectious diseases caused by Salmonellacholeraesuis, Salmonella typhimurium, Salmonella derby, Salmonellainfantis or Salmonella newport, comprising the bacteriophage as anactive ingredient.

Still another object of the present invention is to provide anantibiotic, comprising the bacteriophage as an active ingredient.

Still another object of the present invention is to provide an animalfeed or drinking water comprising the bacteriophage as an activeingredient.

Still another object of the present invention is to provide a sanitizeror cleaner, comprising the bacteriophage as an active ingredient.

Still another object of the present invention is to provide a method forpreventing or treating of infectious diseases caused by Salmonellacholeraesuis, Salmonella typhimurium, Salmonella derby, Salmonellainfantis or Salmonella newport, using the bacteriophage or thecomposition.

Advantageous Effects

The novel bacteriophage of the present invention has a specificbactericidal activity against Salmonella choleraesuis, Salmonellatyphimurium, Salmonella derby, Salmonella infantis or Salmonella newportwith no influences on beneficial bacteria, and excellent acid- andheat-resistance and desiccation tolerance. Therefore, the novelbacteriophage can be used for the prevention or treatment ofsalmonellosis or salmonella food poisoning, which is an infectiousdisease caused by Salmonella choleraesuis, Salmonella typhimurium,Salmonella derby, Salmonella infantis or Salmonella newport, and alsowidely used in animal feeds, drinking water for livestock, sanitizers,and cleaners.

DESCRIPTION OF DRAWINGS

FIG. 1 is an electron microscopy photograph of ΦCJ11, which belongs tothe family Siphoviridae of morphotype B1, characterized by an isometriccapsid and a long non-contractile tail;

FIG. 2 is a photograph showing the formation of ΦCJ11 plaques in a lawnof salmonella bacteria, in which (A,B;SC, C;SG, D;ST, E;SI, F,G;SD,H;SN), plaque formation was observed in lawns of SC, ST, SI, SD and SN,but not in lawns of SG;

FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage ΦCJ11, inwhich the major proteins were detected at 33 kDa, 55 kDa and 69.5 kDa,and Precision plus protein standard (BIO-RAD) was used as a marker;

FIG. 4 is the result of PFGE of the isolated bacteriophage ΦCJ11, inwhich a total genome size of ΦCJ11 was approximately 140 kbp, and theCHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as a DNA sizemarker;

FIG. 5 is the result of PCR, performed using each primer set for theΦCJ11 genomic DNA, in which A; a primer set of SEQ ID NOs. 5 and 6, B; aprimer set of SEQ ID NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and10, and D; a primer set of SEQ ID NOs. 11 and 12, and all of A, B, C andD lanes had PCR products of approximately 1 kbp or more to 2 kbp orless;

FIG. 6 is the result of acid-resistance assay on the bacteriophageΦCJ11, showing the number of surviving bacteriophage at pH 2.1, 2.5,3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0, in which thebacteriophage ΦCJ11 did not lose its activity until pH 5.5, but thebacteriophage ΦCJ11 showed reduced activity at pH 4 and pH 3.5, andcompletely lost its activity at pH 3.0 or lower, as compared to acontrol;

FIG. 7 is the result of heat-resistance assay on the bacteriophageΦCJ11, showing the number of surviving bacteriophage at 37, 45, 53, 60,and 70° C. for 0, 10, 30, 60 and 120 minutes, in which the bacteriophageΦCJ11 maintained its activity even though exposed to 60° C. for up to 2hours;

FIG. 8 is the result of desiccation tolerance assay on the bacteriophageΦCJ11 dried with the aid of a SpeedVac concentrator, in which when titerchanges under the dry condition were measured in comparison withpre-drying titers, the activity was maintained at 60° C. for up to 1hour; and

FIG. 9 is the results of body weight changes due to toxicity aftersingle oral administration of Sprague-Dawley rats with ΦCJ11, in whichobservation of body weight changes before and 1, 3, 7, 10 and 14 daysafter administration with ΦCJ11 showed no significant changes incomparison with the control group (▪; male control, □; ΦCJ11 male, ;female control, ◯; ΦCJ11 female).

BEST MODE

In one aspect to achieve the above objects, the present inventionprovides a novel bacteriophage having a specific bactericidal activityagainst Salmonella choleraesuis, Salmonella typhimurium, Salmonelladerby, Salmonella infantis or Salmonella newport.

The present inventors collected fecal and sewage samples from swinery,and isolated therefrom bacteriophages that can lyse the host cell SC.They were also found that these bacteriophages can lyse ST, SD, SI andSN (FIG. 2 and Table 1). A morphological examination under an electronmicroscope confirmed that the bacteriophage (ΦCJ11) belongs to thefamily Siphoviridae of morphotype B1 (FIG. 1). Further, thebacteriophage ΦCJ11 was found to have major structural proteins ofapproximately 69.5 kDa, 55 kDa and 33 kDa, as measured by a proteinpattern analysis (FIG. 3), and a genome analysis showed that ΦCJ11 has atotal genome size of approximately 97-145.5 kbp (FIG. 4). Furthermore,the results of analyzing its genetic features showed that thebacteriophage includes nucleic acid molecules represented by SEQ ID NOs.1 to 4 within the total genome (Example 6). Based on SEQ ID NOs. 1 to 4,genetic similarity with other species was compared. It was found thatthe bacteriophage showed very low genetic similarity with the knownbacteriophages, indicating that the bacteriophage is a novelbacteriophage (Table 2). For more detail analysis of genetic features,the ΦCJ11-specific primer sets, namely, SEQ ID NOs. 5 and 6, SEQ ID NOs.7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12 were used toperform PCR. Each PCR product was found to have a size of 1.4 kbp, 1.2kbp, 1.25 kbp and 1.5 kbp (FIG. 5).

Meanwhile, when SC, ST, SD, SI and SN were infected with ΦCJ11, thephage plaques (clear zone on soft agar created by host cell lysis of onebacteriophage) were observed (FIG. 2). The stability of ΦCJ11 wasexamined under various temperature and pH conditions, resulting in thatΦCJ11 stably maintains in a wide range of pH environments from pH 3.5 topH 11.0 (FIG. 6) and in high temperature environments from 37° C. to 70°C. (FIG. 7), and even after desiccation (FIG. 8). Also, the wild-typestrains SC, ST, SD, SI and SN were also found to fall within the hostcell range of ΦCJ11 (Table 3).

Finally, the results of dermal and ocular irritation tests on ΦCJ11 inspecific-pathogen-free (SPF) New Zealand White rabbits showed that theprimary irritation index (PII) was 0.33, indicating no irritant, and theindex of acute ocular irritation (IAOI) was 0 in washing and non-washinggroups during the whole experimental periods, indicating no irritant.The results of oral administration of ΦCJ11 showed no changes in weightgain (FIG. 9). As well, mortality, general symptoms (Table 4) and organabnormality (Table 5) were not observed, indicating no toxicity.

Accordingly, the present inventors designated the bacteriophage as“Bacteriophage ΦCJ11”, in which the bacteriophage was isolated fromfecal and sewage samples from swinery and has a specific bactericidalactivity against SC, ST, SD, SI and SN and the above characteristics,and deposited at the Korean Culture Center of Microorganisms (361-221,Honje 1, Seodaemun, Seoul) on Sep. 9, 2011 under accession numberKCCM11208P.

In another aspect to achieve the above objects, the present inventionprovides a composition for the prevention or treatment of infectiousdisease caused by one or more Salmonella bacteria selected from thegroup consisting of Salmonella choleraesuis, Salmonella typhimurium,Salmonella derby, Salmonella infantis and Salmonella newport, comprisingthe bacteriophage as an active ingredient.

Having specific bactericidal activity against Salmonella choleraesuis,Salmonella typhimurium, Salmonella derby, Salmonella infantis andSalmonella newport, the bacteriophage of the present invention may beused for the purpose of preventing or treating the diseases caused bythese bacteria. Preferably, examples of the infectious diseases includeporcine salmonellosis and Salmonella food poisoning caused by Salmonellacholeraesuis or Salmonella typhimurium, and acute or chronic porcineenteritis caused by Salmonella derby, Salmonella infantis, Salmonellanewport, but are not limited thereto.

As used herein, the term “salmonellosis” refers to symptoms followingsalmonella infection, such as fever, headache, diarrhea, and vomiting.That is, salmonellosis is an infection with bacteria of the genusSalmonella, which is defined with two clinical forms: an acutesepticemic form that resembles typhoid fever and an acutegastroenteritis, including enteritis, food poisoning, and acutesepticemia.

As used herein, the term “prevention” means all of the actions in whichdisease progress is restrained or retarded by the administration of thecomposition.

As used herein, the term “treatment” means all of the actions in whichthe condition has taken a turn for the better or been restrained ormodified favorably by the administration of the composition.

The composition of the present invention includes ΦCJ11 in an amount of1×10¹ to 1×10¹² PFU/mL, and preferably in an amount of 1×10⁶ to 1×10¹⁰PFU/mL.

On the other hand, the composition of the present invention may furtherinclude a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. For formulation of the composition into aliquid preparation, a pharmaceutically acceptable carrier which issterile and biocompatible may be used such as saline, sterile water,Ringer's solution, buffered physiological saline, albumin infusionsolution, dextrose solution, maltodextrin solution, glycerol, ethanol,and mixtures of one or more thereof. If necessary, other conventionaladditives such as antioxidants, buffers, and bacteriostatic agents maybe added. Further, diluents, dispersants, surfactants, binders andlubricants may be additionally added to the composition to prepareinjectable formulations such as aqueous solutions, suspensions, andemulsions, or oral formulations such as pills, capsules, granules, ortablets.

The prophylactic or therapeutic compositions of the present inventionmay be applied or sprayed to the afflicted area, or administered by oralor parenteral routes. The parenteral administration may includeintravenous, intraperitoneal, intramuscular, subcutaneous or topicaladministration.

The dosage suitable for applying, spraying, or administrating thecomposition of the present invention will depend upon a variety offactors including formulation method, the mode of administration, theage, weight, sex, condition, and diet of the patient or animal beingtreated, the time of administration, the route of administration, therate of excretion, and reaction sensitivity. A physician or veterinarianhaving ordinary skill in the art can readily determine and prescribe theeffective amount of the composition required.

Examples of the oral dosage forms including the composition of thepresent invention as an active ingredient include tablets, troches,lozenges, aqueous or emulsive suspensions, powder or granules,emulsions, hard or soft capsules, syrups, or elixirs. For formulationsuch as tablets and capsules, the following are useful: a binder such aslactose, saccharose, sorbitol, mannitol, starch, amylopectin, celluloseor gelatin; an excipient such as dicalcium phosphate; a disintegrantsuch as corn starch or sweet potato starch; and a lubricant such asmagnesium stearate, calcium stearate, sodium stearylfumarate, orpolyethylene glycol wax. For capsules, a liquid carrier such as lipidmay be further used in addition to the above-mentioned compounds.

The parenteral dosage forms including the composition of the presentinvention as an active ingredient may be formulated into injections viasubcutaneous, intravenous, or intramuscular routes, suppositories, orsprays inhalable via the respiratory tract, such as aerosols. Injectionforms may be prepared by dissolving or suspending the composition of thepresent invention, together with a stabilizer or a buffer, in water andloading the solution or suspension onto ampules or vial unit forms. Forsprays, such as aerosols, a propellant for spraying a water-dispersedconcentrate or wetting powder may be used in combination with anadditive.

In still another aspect to achieve the above objects, the presentinvention provides an antibiotic comprising the bacteriophage as anactive ingredient.

As used herein, the term “antibiotic” means any drug that is applied toanimals to kill pathogens, and used herein as a general term forantiseptics, bactericidal agents and antibacterial agents. The animalsare mammals including human. The bacteriophage of the present invention,unlike the conventional antibiotics, has a high specificity toSalmonella so as to kill the specific pathogens without affectingbeneficial bacteria, and does not induce drug resistance so that it canbe provided as a novel antibiotic with a comparatively long life cycle.

In still another aspect to achieve the above objects, the presentinvention provides an animal feed or drinking water comprising thebacteriophage as an active ingredient.

In-feed antibiotics used in the livestock and fishery industries areintended to prevent infections. However, most of the currently availablein-feed antibiotics are problematic in that they are apt to induce theoccurrence of resistant strains and may be transferred to humans, due toremaining in livestock products. The uptake of such residual antibioticsmay make human pathogens resistant to antibiotics, resulting in thespread of diseases. In addition, since there are a variety of in-feedantibiotics, the increasing global emergence of multidrug-resistantstrain is a serious concern. Therefore, the bacteriophage of the presentinvention can be used as an in-feed antibiotic that is more eco-friendlyand able to solve the above problems.

The animal feed of the present invention may be prepared by adding thebacteriophage directly or in separate feed additive form to an animalfeed. The bacteriophage of the present invention may be contained in theanimal feed as a liquid or in a solid form, preferably in a driedpowder. The drying process may be performed by air drying, naturaldrying, spray drying, and freeze-drying, but is not limited thereto. Thebacteriophage of the present invention may be added as a powder form inan amount of 0.05 to 10% by weight, preferably 0.1 to 2% by weight,based on the weight of animal feed. The animal feed may also includeother conventional additives for long-term preservation, in addition tothe bacteriophage of the present invention.

The feed additive of the present invention may additionally includeother non-pathogenic microorganisms. The available additionalmicroorganism may be selected from the group consisting of Bacillussubtilis that can produce protease, lipase and invertase, Lactobacillussp. strain that can exert physiological activity and a function ofdecomposing under anaerobic conditions, such as in the stomach ofcattle, filamentous fungi including Aspergillus oryzae (J Animal Sci43:910-926, 1976) that increases the weight of domestic animals,enhances milk production and helps the digestion and absorptiveness offeeds, and yeast including Saccharomyce scerevisiae (J Anim Sci56:735-739, 1983).

The feed including ΦCJ11 of the present invention may includeplant-based feeds, such as grain, nut, food byproduct, seaweed, fiber,drug byproduct, oil, starch, meal, and grain byproduct, and animal-basedfeeds such as protein, inorganic matter, fat, mineral, fat, single cellprotein, zooplankton, and food waste, but is not limited thereto.

The feed additive including ΦCJ11 of the present invention may includebinders, emulsifiers, and preservatives for the prevention of qualitydeterioration, amino acids, vitamins, enzymes, probiotics, flavorings,non-protein nitrogen, silicates, buffering agents, coloring agents,extracts, and oligosaccharides for efficiency improvement, and otherfeed premixtures, but is not limited thereto.

Further, the supply of drinking water mixed with the bacteriophage ofthe present invention can reduce the number of Salmonella bacteria inthe intestine of livestock, thereby obtaining Salmonella-free livestock.

In still another aspect to achieve the above objects, the presentinvention provides a sanitizer or cleaner comprising the bacteriophageas an active ingredient.

In still another aspect to achieve the above objects, the presentinvention provides a method for treating infectious diseases caused bySalmonella choleraesuis, Salmonella typhimurium, Salmonella derby,Salmonella infantis or Salmonella newport using the bacteriophage or thecomposition.

In detail, the therapeutic method of the present invention comprises thestep of administering a pharmaceutically effective amount of thebacteriophage or the composition to an individual having infectiousdiseases caused by Salmonella choleraesuis, Salmonella typhimurium,Salmonella derby, Salmonella infantis or Salmonella newport.

The bacteriophage or the composition of the present invention may beadministered in the form of a pharmaceutical formulation into animals ormay be ingested as a mixture with animal feed or drinking water byanimals and preferably as a mixture with animal feed.

As long as it reaches target tissues, any route, whether oral orparenteral, may be taken for administering the bacteriophage or thecomposition of the present invention. In detail, the composition of thepresent invention may be administered in a typical manner via any routesuch as oral, rectal, topical, intravenous, intraperitoneal,intramuscular, intraarterial, transdermal, intranasal, and inhalationroutes.

It will be obvious to those skilled in the art that the total daily doseof the bacteriophage or the composition of the present invention to beadministered by the therapeutic method should be determined throughappropriate medical judgment by a physician. Preferably, thetherapeutically effective amount for given patients may vary dependingon various factors well known in the medical art, including the kind anddegree of the response to be achieved, the patient's condition such asage, body weight, state of health, sex, and diet, time and route ofadministration, the secretion rate of the composition, the time periodof therapy, concrete compositions according to whether other agents areused therewith or not, etc.

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detail withreference to Examples. However, these Examples are for illustrativepurposes only, and the invention is not intended to be limited by theseExamples.

Example 1 Salmonella Bacteriophage Isolation Example 1-1 BacteriophageScreening and Single Bacteriophage Isolation

50 mL of sample from swinery and sewage effluent was transferred to acentrifuge tube, and centrifuged at 4000 rpm for 10 minutes. Then, thesupernatant was filtered using a 0.45 μm filter. 18 mL of samplefiltrate was mixed with 150 μl of Salmonella choleraesuis (“SC”) shakingculture medium (OD₆₀₀=2) and 2 mL of 10× Luria-Bertani medium (tryptone10 g/L, yeast extract 5 g/L and NaCl 10 g/L: LB medium). The mixture wascultured at 37° C. for 18 hours, and the culture medium was centrifugedat 4000 rpm for 10 minutes. The supernatant was filtered using a 0.2 μmfilter. 3 mL of 0.7% agar (w/v) and 150 μl of SC shaking culture medium(OD₆₀₀=2) were mixed, and plated onto LB plate (“top-agar”), and allowedto solidify. 10 μl of the culture filtrate was spread thereon, andcultured for 18 hours at 37° C., and the titration of phage lysate wasperformed on the top-agar, called soft agar overlay method.

The sample culture medium containing the phage lysate was properlydiluted, and mixed with 150 μl of SC shaking culture medium (OD₆₀₀=2),followed by soft agar overlay method, so that single plaques wereobtained. A single plaque represents one bacteriophage and thus, forisolation of single bacteriophages, one phage plaque was added to 400 μlof SM solution (NaCl, 5.8 g/L, MgSO₄7H₂O, 2 g/L, 1 M Tris-Cl (pH 7.5) 50ml/L), and left for 4 hours at room temperature to isolate singlebacteriophages. To purify the bacteriophage in large quantities, 100 μlof supernatant was taken from the single bacteriophage solution, andmixed with 12 mL of 0.7% agar and 500 μl of SC shaking culture medium,followed by soft agar overlay method on LB plate having a diameter of150 mm. When lysis was completed, 15 mL of SM solution was added to theplate. The plate was gently shaken for 4 hours at room temperature toelute the bacteriophages from the top-agar. The SM solution containingthe eluted bacteriophages was recovered, chloroform was added to a finalvolume of 1%, and mixed well for 10 minutes. The solution wascentrifuged at 4000 rpm for 10 minutes. The obtained supernatant wasfiltered using a 0.45 μm filter, and stored in the refrigerator.

Example 1-2 Large-Scale Batches of Bacteriophage

The selected bacteriophages were cultured in large quantities using SC.SC was shaking-cultured, and an aliquot of 1.5×10¹⁰ cfu (colony formingunits) was centrifuged at 4000 rpm for 10 minutes, and the pellet wasresuspended in 4 ml of SM solution. The bacteriophage of 9.0×10⁸ PFU(plaque forming unit) was inoculated thereto (MOI: multiplicity ofinfection=0.001), and left at 37° C. for 20 minutes. The solution wasinoculated into 150 ml of LB media, and cultured at 37° C. for 5 hours.Chloroform was added to a final volume of 1%, and the culture solutionwas shaken for 20 minutes. DNase I and RNase A were added to a finalconcentration of 1 μg/ml, respectively. The solution was left at 37° C.for 30 minutes. NaCl and PEG (polyethylene glycol) were added to a finalconcentration of 1 M and 10% (w/v), respectively and left at 4° C. foran additional 3 hours. The solution was centrifuged at 4° C. and 12,000rpm for 20 minutes to discard the supernatant. The pellet wasresuspended in 5 mL of SM solution, and left at room temperature for 20minutes. 4 mL of chloroform was added thereto and mixed well, followedby centrifugation at 4° C. and 4000 rpm for 20 minutes. The supernatantwas filtered using a 0.2 μm filter, and the bacteriophage was purifiedby glycerol density gradient ultracentrifugation (density: 40%, 5%glycerol at 35,000 rpm and 4° C. for 1 hour). The purified bacteriophagewas designated as “Bacteriophage ΦCJ11”, and resuspended in 300 μl of SMsolution, followed by titration. The bacteriophage φCJ11 was depositedat the Korean Culture Center of Microorganisms (361-221, Honje 1,Seodaemun, Seoul) on Sep. 9, 2011 under accession number KCCM11208P.

Example 2 Examination on φCJ11 Infection of Salmonella

To analyze the selected bacteriophage for lytic activity on Salmonellaspecies other than SC, attempts were made of cross infection with otherSalmonella species. As a result, ΦCJ11 infected SC (Salmonellacholeraesuis), ST (Salmonella typhimurium), SD (Salmonella derby), SN(Salmonella newport), SI (Salmonella infantis), SA (Salmonella arizonae)and SB (Salmonella bongori), but did not infect SE (Salmonellaenteritidis), SG (Salmonella gallinarum), and SP (Salmonella pullorum)(Table 1 and FIG. 2).

TABLE 1 ΦCJ11 Infection of Salmonella Phage Phage Sero- plaque Sero-plaque type Strain name formation type Strain name formation SC ATCC10708 ◯ SA ATCC 12398 ◯ SN SL 317 ◯ SB ATCC 12397 ◯ SD ATCC 2468 ◯ ST 13◯ SE SGSC 2282 X SI SARB 26 ◯ SG SGSC 2293 X SP SGSC 2295 X * ATCC:American Type Culture Collection * SGSC: Salmonella Genetic Stock Center

Moreover, FIG. 2 is a photograph showing the formation of ΦCJ11 plaquesin a lawn of salmonella bacteria. As shown in FIG. 2 (A,B;SC, C;SG,D;ST, E;SI, F,G;SD, H;SN), plaque formation was observed in lawns of SC,ST, SI, SD and SN, but not in lawns of SG.

Example 3 Morphology of #CJ11

The purified ΦCJ11 was diluted in the SM buffer solution, and thenmounted on a copper grid, stained with 2% uranyl acetate for 3 to 5seconds, and dried. Examination under a transmission electron microscope(LIBRA 120, Carl Zeiss transmission electron Microscope, 80 kV,magnification of ×120,000˜×200,000) was performed (FIG. 1). FIG. 1 is anelectron microscopy photograph of ΦCJ11. As shown in FIG. 1, it wasfound that the purified ΦCJ11 belongs to the family Siphoviridae ofmorphotype B1, characterized by an isometric capsid and a longnon-contractile tail.

Example 4 Protein Pattern Analysis of ΦCJ11

15 μL of a ΦCJ11 solution purified at a titer of 10¹¹ PFU/mL was mixedwith 3 μL of a 5×SDS sample solution, and heated for 5 minutes. 12%SDS-PAGE was performed, and then the gel was stained with Coomassie bluefor 1 hour at room temperature (FIG. 3). FIG. 3 is the result ofSDS-PAGE of the isolated bacteriophage ΦCJ11, in which Precision plusprotein standard (BIO-RAD) was used as a marker. As shown in FIG. 3, themajor proteins were detected at 33 kDa, 55 kDa and 69.5 kDa.

Example 5 Analysis of Total Genomic DNA Size of ΦCJ11

Genomic DNA of the purified ΦCJ11 was isolated usingultracentrifugation. In detail, to the purified ΦCJ11 culture mediumwere added EDTA (ethylenediaminetetraacetic acid (pH 8.0)), proteinaseK, and SDS (sodium dodecyl sulfate) at a final concentration of 20 mM,50 μg/mL, and 0.5% (w/v), respectively, followed by incubation at 50° C.for 1 hour. An equal volume of phenol (pH 8.0) was added and mixed well.After centrifugation at room temperature and 12,000 rpm for 10 minutes,the supernatant was mixed well with an equal volume of PC(phenol:chloroform=1:1). Another centrifugation was performed at roomtemperature and 12,000 rpm for 10 minutes. Then, a supernatant wasobtained, and mixed with an equal volume of chloroform, followed bycentrifugation at room temperature and 12,000 rpm for 10 minutes. Theobtained supernatant was mixed with 1/10 volume of 3 M sodium acetateand two volumes of cold 95% ethanol, and left at −20° C. for 1 hour.After centrifugation at 0° C. and 12,000 rpm for 10 minutes, thesupernatant was completely removed, and the DNA pellet was dissolved in50 μL of TE (Tris-EDTA (pH 8.0)). The extracted DNA was diluted 10-fold,and measured for absorbance at OD₂₆₀ to determine its concentration. 1μg of the total genomic DNA was loaded onto 1% PFGE (pulse-field gelelectrophoresis) agarose gel, and electrophoresed at 14° C. for 22 hoursin a BIORAD CHEF DR II PFGE system under the conditions of switch timeramp for 50-90 seconds, 6 V/cm (200V). The CHEF DNA Size Standard LambdaLadder (Bio-Rad) was used as a DNA size marker (FIG. 4). FIG. 4 is theresult of PFGE of the isolated bacteriophage ΦCJ11. As shown in FIG. 4,DNA of approximately 140 kbp present between 48.5 to 1,000 kbp wasobserved.

Example 6 Genetic Analysis of ΦCJ11

For the genetic analysis of the purified ΦCJ11, 5 μg of the genomic DNAof ΦCJ11 was double digested with the restriction enzymes PstI, XbaI andBamHI, EcoRI and SalI. The vector pCL1920 (Promega) was digested withthe restriction enzymes PstI, XbaI and BamHI, EcoRI and SalI, and thentreated with CIP (calf intestinal alkaline phosphatase). The digestedgenomic DNA was mixed at a ratio of 3:1 with the vector, and ligated at16° C. for 2 hours. The resulting recombinant vector was transformedinto E. coli DH5a which was then plated on an LB plate containingspecinomycin and X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for selection ofblue/white colonies. The selected colonies were cultured for 16 hours ina culture medium containing the antibiotic with shaking. Then, plasmidswere extracted using a Plasmid purification kit (Promega).

The cloning of the plasmids was confirmed by PCR using primer sets ofFTR135 and FTR136 (SEQ ID NOs. 13 and 14) and selection was made only ofinsert fragments having a size of 1 kb or longer. Their nucleotidesequences were analyzed using the primer sets. The nucleotide sequencesthus obtained were given in SEQ ID NOs. 1 to 4, respectively, eachhaving a size of 1 to 2 kbp or less, and analyzed for sequencesimilarity with the aid of NCBI blastx and blastn program, and theresults are summarized in Table 2, below.

TABLE 2 Comparison of Sequence Similarity between ΦCJ11 and OtherBacteriophages Blastx Organism Protein Query Subject Identity e-value 1Enterobacteria hypothetical protein 436-627 1-64  63/64 7e−32 phage T5(98%) Enterobacteria hypothetical protein  10-423 1-139 127/139 3e−31phage SPC35 (91%) Enterobacteria hypothetical protein  3-863 30-315 264/291  2e−116 phage SPC35 (91%) Enterobacteria hypothetical protein 3-863 30-315  255/291  1e−113 phage T5 (88%) Enterobacteriahypothetical protein  3-761 29-281  235/253  7e−107 phage EPS7 (93%)Klebsiella hypothetical protein  9-761 22-276  217/255  4e−100 phageKP15 (85%) Enterobacteria putative SPFH domain-  9-839 18-257  223/2817e−99 phage RB43 containing protein (79%) Enterobacteria hypotheticalprotein  9-761 18-272  219/255 2e−98 phage RB16 (86%) 2 Enterobacteriahypothetical protein 412-771 1-120 109/120 7e−57 phage EPS7 (91%)Enterobacteria hypothetical protein 490-771 1-94  94/94 3e−46 phage T5(100%)  Enterobacteria D11 protein 532-2  1-177 175/177 5e−96 phage T5(99%) Enterobacteria D11 protein 532-2  1-177 174/177 2e−95 phage SPC35(98%) Enterobacteria D11 protein 532-8  1-175 163/175 4e−90 phage EPS7(93%) Enterobacteria hypothetical protein 872-528 5-120  96/116 2e−46phage EPS7 (83%) 3 Enterobacteria tail protein Pb4 777-4  1-258 221/258 4e−128 phage SPC35 (86%) Enterobacteria tail protein Pb4 777-4  1-258197/258  6e−115 phage T5 (76%) Enterobacteria tail protein Pb3 1322-780 769-949  178/181 2e−96 phage SPC35 (98%) Enterobacteria tail protein Pb31322-780  769-949  160/181 9e−88 phage EPS7 (88%) Enterobacteriastructural tail 1322-780  769-949  156/181 8e−84 phage T5 protein Pb3(86%) Enterobacteria tail protein Pb4 423-4  1-140 112/140 2e−62 phageEPS7 (80%) 4 Enterobacteria flap endonuclease 980-564 153-291  138/1391e−73 phage SPC35 (99%) Enterobacteria flap endonuclease 980-564153-291  138/139 1e−73 phage T5 (99%) Enterobacteria flap endonuclease980-564 153-291  135/139 4e−72 phage EPS7 (97%) Enterobacteria putativedeoxyUTP 564-157 1-136 130/136 3e−70 phage SPC35 pyrophosphatase (96%)Enterobacteria putative deoxyUTP 564-160 1-135 130/135 2e−69 phage T5pyrophosphatase (96%) Enterobacteria putative deoxyUTP 564-157 1-136128/136 2e−67 phage EPS7 pyrophosphatase (94%)

Example 7 Design of ΦCJ11-Specific Primer Sequences

In order to identify ΦCJ11, ΦCJ11-specific primers were designed on thebasis of SEQ ID NOS. 1 to 4. PCR was performed using each primer set ofSEQ ID NOS. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQID NOs. 11 and 12. 0.1 μg of the genomic DNA of bacteriophage and 0.5pmol of each primer were added to a pre-mix (Bioneer), and the finalvolume was adjusted to 20 μL. PCR was performed with 30 cycles ofdenaturation; 94° C. 30 seconds, annealing; 55° C. 30 seconds, andpolymerization; 72° C., 1.5 minutes (FIG. 5). FIG. 5 is the result ofPCR, performed using each primer set for the ΦCJ11 genomic DNA. A; aprimer set of SEQ ID NOs. 5 and 6, B; a primer set of SEQ ID NOs. 7 and8, C; a primer set of SEQ ID NOs. 9 and 10, and D; a primer set of SEQID NOs. 11 and 12. All of A, B, C and D lanes had PCR products ofapproximately 1 to 2 kbp. As shown in FIG. 5, the PCR products thusobtained had a size of approximately 1 kbp or more to 2 kbp or less,with the primer sets of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ IDNOs. 9 and 10, and SEQ ID NOs. 11 and 12.

Example 8 pH Stability of Bacteriophage

In order to determine whether ΦCJ11 survives with stability under thelow pH environment in the stomach of pig, ΦCJ11 was assayed forstability in a wide range of pH (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4,6.9, 7.4, 8.2, 9.0, 9.8 and 11.0). Various pH solutions (sodium acetatebuffer (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer (pH2.5, pH 3.0, and pH 3.5), sodium phosphate buffer (pH 6.9 and pH 7.4)and Tris-HCl (pH 8.2, pH 9.0, pH 9.8 and pH 11.0)) were prepared to havea concentration of 0.2 M. 180 μL of each pH solution was mixed with 20μL of a bacteriophage solution (1.0×10¹¹ PFU/mL) to give each pHsolution a concentration of 1 M, followed by incubation at roomtemperature for 2 hours. The reaction solution was serially diluted, and10 μL of each dilution was cultured at 37° C. for 18 hours by a softagar overlay method to determine the titers of the phage lysates (FIG.6). FIG. 6 is the result of acid-resistance assay on the bacteriophageΦCJ11, showing the number of surviving bacteriophage at pH 2.1, 2.5,3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0. Thebacteriophage ΦCJ11 did not lose its activity until pH 5.5. However, thebacteriophage ΦCJ11 showed reduced activity at pH 4 and pH 3.5, andcompletely lost its activity at pH 3.0 or lower, as compared to acontrol. As shown in FIG. 6, the bacteriophage did not lose its activityand remained stable down to pH 5.5 whereas it lost its activity at pH3.0 or lower.

Example 9 Heat Stability of Bacteriophage

For use as a feed additive, the bacteriophage was assayed for stabilityto the heat generated during a formulation process. In this regard, 200μL of a ΦCJ11 solution with a titer of 1.0×10¹¹ PFU/mL was incubated at37° C., 45° C., 53° C., 60° C., and 70° C. for 0 minute, 10 minutes, 30minutes, 60 minutes and 120 minutes. The solution was serially diluted,and 10 μL of each dilution was cultured at 37° C. for 18 hours by a softagar overlay method to determine the titers of phage lysates (FIG. 7).FIG. 7 is the result of heat-resistance assay on the bacteriophageΦCJ11, showing the number of surviving bacteriophage at 37° C., 45° C.,53° C., 60° C., and 70° C. for 0, 10, 30, 60 and 120 minutes. As shownin FIG. 7, the bacteriophage ΦCJ11 maintained its activity even thoughexposed at 60° C. up to 2 hours.

Example 10 Desiccation Tolerance of Bacteriophage

For use as a feed additive, the bacteriophage ΦCJ11 was assayed fortolerance to the dry condition set for a formulation process. On thebasis of the results obtained from the heat stability assay, adesiccation assay was performed using a SpeedVac concentrator. 200 μL ofa ΦCJ11 solution having a titer of 1.0×10¹¹ PFU/mL was dried undervacuum at 60° C. for 2 hours, and the pellet thus obtained wascompletely re-suspended in 200 μL of the SM solution at 4° C. for oneday, and measured for titer values (FIG. 8). FIG. 8 is the result ofdesiccation tolerance assay on the bacteriophage ΦCJ11 dried with theaid of a SpeedVac concentrator. As shown in FIG. 8, when titer changesunder the dry condition were measured in comparison with pre-dryingtiters, the activity was maintained at 60° C. up to 1 hour.

Example 11 Infection Spectrum of Bacteriophage

ΦCJ11 was assayed for lytic activity against the wild-type (2 strains),Salmonella choleraesuis (5 strains), Salmonella typhimurium (17strains), Salmonella infantis (4 strains), Salmonella newport (6strains), Salmonella derby (2 strains) and Salmonella dublin (3strains), obtained from Laboratory of Avian Diseases, College ofVeterinary Medicine, Seoul National University, in addition to SC (ATCCSC10708) used in the experiment. 150 μL of each strain shaking culturemedium (OD₆₀₀=2) was mixed, and 10 μL of ΦCJ11 solution having a titerof 10¹⁰ PFU/mL was cultured at 37° C. for 18 hours using a soft agaroverlay method to monitor the was observed in 8 strains of SC.

TABLE 3 Wild-type strains SC, ST, SD, SI, SN infected by ΦCJ11 PhagePhage plaque plaque Serotype Strain name formation serotype Strain nameformation SC S. choleraesuis ATCC ∘ SI S. infantis SARB 26 ∘ 2929 S.choleraesuis ATCC ∘ S. infantis SARB 27 ∘ 2930 S. choleraesuis ATCC ∘ S.infantis S1326/28 ∘ 2932 S. choleraesuis ATCC ∘ S. infantis B09-106 ∘2933 S. choleraesuis ATCC ∘ SN S. newport SARB 36 ∘ 2425 S. choleraesuisATCC ∘ S. newport SARB 37 ∘ 10708 S. choleraesuis SNU#1 ∘ S. newportSARB 38 ∘ S. choleraesuis SNU#2 ∘ S. newport 7257 ∘ ST S. typhimuriumSNU ∘ S. newport SL 317 ∘ ST1 S. typhimurium SNU ∘ S. newport SL 254 ∘ST2 S. typhimurium SNU ∘ SD S. derby ATCC ∘ ST4 2466 S. typhimurium SNU∘ S. derby ATCC ∘ ST7 2468 S. typhimurium SNU ∘ SD S. dublin SA 4405 ∘ST8 S. typhimurium SNU ∘ S. dublin RKS 4699 ∘ ST11 S. typhimurium SNU ∘S. dublin 88/6 ∘ ST12 S. typhimurium SNU ∘ SA S. arizonae ATCC ∘ ST1312398 S. typhimurium SNU ∘ SB S. bongori ATCC ∘ ST14 12397 S.typhimurium SNU ∘ SH S. heidelberg SARA 33 ∘ ST17 S. typhimurium SNU ∘S. heidelberg SARA 23 ∘ ST18 S. typhimurium SNU ∘ SM S. maimi SARB 28 ∘ST19 S. typhimurium SNU ∘ S. maimi SARB 29 ∘ ST20 S. typhimurium SNU ∘SP S. panama SARB 39 ∘ ST26 S. typhimurium SNU ∘ S. panama SARB 40 ∘ST38 S. typhimurium SNU ∘ S. panama SARB 41 ∘ ST41 S. typhimurium SNU ∘S. panama 7261 ∘ ST42 * SGSC: Salmonella genetic stock center * ATCC:American Type Culture Collection * SNU: Laboratory of Avian Diseases,College of Veterinary Medicine, Seoul National University

Example 12 Toxicity Assay of Bacteriophage

Dermal and ocular irritation tests were performed inspecific-pathogen-free (SPF) New Zealand white rabbits, which arecommonly used in the toxicity test of bacteriophage ΦCJ11 for theprevention of salmonellosis and salmonella food poisoning, and of whichexperimental data were accumulated to allow easy analysis of experimentresults. The normal abdominal skin (non-injured skin) and injuredabdominal skin of rabbits were covered and contacted with 2.5 cm×2.5 cmof gauze applied with the test substance, and each 0.5 mL/site wasapplied. No changes in general symptoms were observed, and a slightweight loss was observed 1 day after application of the test substance,which can likely to be attributed to stress due to occlusive applicationof the test substance. In the dermal irritation test, the primaryirritation index (PII) was 0.33, indicating no irritant. For the ocularirritation test, the left eye of a rabbit was applied with the testsubstance, and then compared to the right eye, which was not appliedwith the test substance. During the experimental period, generalsymptoms and abnormal changes in body weight related to application ofthe test substance were not observed. After application of the testsubstance, the eye examination showed that the index of acute ocularirritation (IAOI) was “0”, indicating no irritant. Therefore, theseresults indicate that the novel bacteriophage ΦCJ11 has no toxicity.

Further, toxicity assay was performed by single oral administration ofSprague-Dawley rats with ΦCJ11. A test substance-administered grouptreated with 1×10¹¹ PFU/kg of ΦCJ11 and an excipient control grouptreated with a vehicle [20 mM Tris-HCl (pH 7.0)+2 mM MgCl] as anexcipient were prepared, and 10 rats of each group (5 each of female andmale sexes) were orally administered with a single dosage. Mortality,general symptoms, changes in body weight, and autopsy findings weremonitored for 2 weeks and compared to each other. Monitoring wasconducted every 6 hours, starting from 30 minutes to 1 hour afteradministration on the day of administration. Then, general symptoms weremonitored once a day for 14 days, and recorded thereof (Tables 4 and 5).

TABLE 4 Mortality and general symptoms after oral administration ofΦCJ11 Done Day after treatment Sex (pfu) 1 2 3 4 5 6 7 8 9 10 11 12 1314 Mortality Male Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10¹¹ 0 0 0 0 0 00 0 0 0 0 0 0 0 0 Female Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10¹¹ 0 00 0 0 0 0 0 0 0 0 0 0 0 0

TABLE 5 Autopsy findings after oral administration of ΦCJ11 Sex Done(PFU) gross finding Frequency^(A) Male Control No gross finding 5/5 10¹¹No gross finding 5/5 Female Control No gross finding 5/5 10¹¹ No grossfinding 5/5 ^(A)Number of animals with the sign/number of animalsexamined.

As shown in Tables 4 and 5, none of them died, and neither toxicsymptoms nor noticeable clinical symptoms were generated by ΦCJ11. Theresults are summarized in Tables 4 and 5. Body weights were recordedbefore administration and 1, 3, 7, 10 and 14 days after administration.No significant changes were observed in body weight compared to thecontrol group.

Meanwhile, the results of body weight changes indicate that ΦCJ11 doesnot cause a toxic reaction sufficient to reduce appetite or to changebody weight. These results are shown in FIG. 9. FIG. 9 is the results ofbody weight changes due to toxicity after single oral administration ofSprague-Dawley rats with ΦCJ11. As shown in FIG. 9, observation of bodyweight changes before administration and 1, 3, 7, 10 and 14 days afteradministration with ΦCJ11 showed that no significant changes in bodyweight were found in comparison with the control group (▪; male control,□; ΦCJ11 male, ; female control, ◯; ΦCJ11 female).

Therefore, it was found that ADL of the novel bacteriophage ΦCJ11exceeds 1×10¹¹ PFU/kg in both female and male rats, and thus it isnon-toxic.

1. An isolated bacteriophage having a specific bactericidal activityagainst Salmonella choleraesuis, which is identified by accession numberKCCM11208P.
 2. A composition for the prevention or treatment ofinfectious diseases caused by Salmonella bacteria selected from thegroup consisting of Salmonella choleraesuis, Salmonella typhimurium,Salmonella derby, Salmonella infantis, Salmonella newport andcombinations thereof, comprising the bacteriophage of claim 1 as anactive ingredient.
 3. The composition according to claim 2, wherein theinfectious disease caused by Salmonella choleraesuis or Salmonellatyphimurium is salmonellosis or Salmonella food poisoning, and theinfectious disease caused by Salmonella derby, Salmonella infantis andSalmonella newport is bacterial infection-type Salmonella foodpoisoning.
 4. An antibiotic, comprising the bacteriophage of claim 1 asan active ingredient.
 5. An animal feed or drinking water, comprisingthe bacteriophage of claim 1 as an active ingredient.
 6. A sanitizer orcleaner, comprising the bacteriophage of claim 1 as an activeingredient.
 7. A method for preventing or treating infectious diseasescaused by one or more Salmonella bacteria selected from the groupconsisting of Salmonella choleraesuis, Salmonella typhimurium,Salmonella derby, Salmonella infantis, Salmonella newport andcombinations thereof, comprising administering the bacteriophage ofclaim 1 to animals in need thereof.
 8. A method for preventing ortreating infectious diseases caused by one or more Salmonella bacteriaselected from the group consisting of Salmonella choleraesuis,Salmonella typhimurium, Salmonella derby, Salmonella infantis,Salmonella newport and combinations thereof, comprising administeringthe composition of claim 2 to animals in need thereof.